Vegf-binding protein for blockade of angiogenesis

ABSTRACT

Provided are chimeric VEGF-binding proteins and nucleic acids (e.g., a vector) encoding chimeric VEGF-binding proteins, methods and host cells for producing these proteins and nucleic acids, and pharmaceutical compositions containing these proteins and nucleic acids. Also provided are methods of treating an angiogenic disease or disorder that include administering at least one of the chimeric VEGF-binding proteins or at least one of the nucleic acids (e.g., a vector) encoding a chimeric VEGF-bind ing protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Application No. 61/525,278,filed on Aug. 19, 2011, the entire contents of which is herebyincorporated by reference.

FIELD OF INVENTION

The present invention is related to the treatment ofangiogenesis-related diseases and disorders.

BACKGROUND OF INVENTION

Vascular endothelial growth factor (VEGF) is an endothelialcell-specific mitogenic and angiogenic inducer that mediates its effectthrough at least two high affinity-binding receptor tyrosine kinases,Flt-1 and KDR, which are expressed only on the surface of vascularendothelial cells. Flt-1 is required for endothelial cell morphorgenesiswhereas KDR is involved primarily with mitogenesis. Gene knockoutstudies have shown that both Flt-1 and KDR are essential for the normaldevelopment of the mammalian vascular system despite their distinctrespective roles in endothelial cell proliferation and differentiation.Both Flt-1 and KDR tyrosine kinase receptors have sevenimmunoglobulin-like (Ig-like) domains which form the extracellularligand-binding regions of the receptors, a transmembrane domain, and anintracellular catalytic tyrosine kinase domain.

VEGF plays a critical role during normal embryonic angiogenesis and alsoin the pathological development of new blood vessels in a number ofdiseases including cancer and in aberrant angiogenesis such ashemangiomas. Solid tumors use blood vessels to obtain oxygen andnutrients and to remove waste materials. In addition these tumorsproduce stromal factors that induce the formation of new blood vesselsto support the tumors' continued growth. Therefore an anti-angiogenicapproach represents an attractive and feasible cancer therapeuticoption, for example, by inhibiting the VEGF signaling pathway.

One strategy of blocking the VEGF signaling pathway is to sequester awaycirculating serum VEGF using VEGF binding proteins. VEGF bindingproteins serve as decoy receptors working to reduce the amount ofcirculating VEGF ligand. VEGF binding proteins include e.g., humanizedmonoclonal antibodies, soluble VEGF receptors, or chimeric VEGF-trapmolecules. Chimeric VEGF traps containing anywhere from two to seven ofthe extracellular Ig-like domains in various combinations have beendescribed (Holash J. et al. Proc. Natl. Acad. Sci. USA 2002,99:11393-98; Davis-Smyth T. et. al. EMBO 1996, 15:4919-4927; U.S. Pat.No. 6,100,071; U.S. Pat. No. 7,087,411), these VEGF-traps are notoptimally effective because they vary in their molecular sizes,VEGF-binding affinity, anti-tumor activity, and pharmacokinetics invivo. These VEGF-traps are currently administered systemically, andmultiple administrations are required in order to maintain a sustaineddelivery for the VEGF-traps to be therapeutically effective. Agene-therapy approach for sustained delivery of VEGF-traps has beenattempted using adenoviruses but the method has been hampered by tissuetoxicity issues and low expression of the transgene.

SUMMARY OF THE INVENTION

Embodiments of the present invention are based on the discovery that achimeric protein comprising a single immunoglobulin-like domain(Ig-like) derived from the vascular endothelial growth factor receptortyrosine kinase Flt-1 and the fragment crystallizable (Fc) constantregion of an immunoglobulin has potent anti-tumor activity in multipletumor models. Such chimeric VEGF-binding protein effectively inhibitstumor growth in vivo. In a particular embodiment, the chimericVEGF-binding protein can be effectively delivered via intramuscularinjection of adeno-associated virus expressing the protein.

In one aspect, a chimeric VEGF-binding protein comprising a firstportion and second portion is provided. In one embodiment of thisaspect, the first portion can consist of, for example, amino acids129-231 of the flt-1 tyrosine kinase receptor (Genbank Accesion No.:BCO39007, SEQ ID NO: 1), while the second portion comprises an Fc regionof immunoglobulin G1 (e.g., amino acids 247-473 of Genbank accession #BC092518 or amino acids 243-469 of SEQ ID NO: 2).

In another embodiment, the chimeric VEGF-binding protein can furthercomprise a signal peptide, including, but not limited to the signalpeptide of the flt-1 tyrosine kinase receptor (SEQ ID NO:5).

In another embodiment, the Fc region of immunoglobulin G1 carried by thechimeric polypeptide is a human Fc region of immunoglobulin G1. Forexample, the Fc region of immunoglobulin G1 can comprise or consist ofamino acids 247-473 of IgG1 (Genbank Accession No.: BC092518). Inanother example, the Fc region of immunoglobulin G1 can comprise orconsist of amino acids 243-469 of SEQ ID NO: 2.

In another embodiment, the Fc region can comprise a reduced immunogenicderivative of an Fc region of immunoglobulin G1.

In another aspect, the invention provides a pharmaceutical compositioncomprising a chimeric VEGF-binding protein of the first aspect and apharmaceutically acceptable carrier.

In another aspect, the invention provides an isolated polynucleotideencoding a chimeric VEGF-binding protein of the first aspect. In oneembodiment, the polynucleotide can be in the form of a recombinantvector comprising the polynucleotide encoding a chimeric VEGF-bindingprotein of the first aspect. In another embodiment, the recombinantvector can be an expression vector that is, e.g., compatible with aprotein expression system using host cells including mammalian cells,insect cells, yeast cells, bacterial cells and plant cells. Theinvention also provides a host cell comprising such a recombinantexpression vector. In one embodiment, the vector is a viral vector,including, but not limited to an adeno-associated virus (AAV) vector ora lentivial vector.

In another aspect, provided are methods of treating an angiogenicdisease or disorder, the methods comprising administering to a subjectin need thereof a vector comprising an isolated polynucleotide encodinga chimeric VEGF-binding protein of the first aspect or administering apharmaceutical composition comprising a chimeric VEGF-binding protein ofthe first aspect and a pharmaceutically acceptable carrier.

In one embodiment of this aspect, provided are methods of treating anangiogenic disease or disorder, the methods comprising administering toa subject in need thereof a pharmaceutical composition comprising achimeric VEGF-binding protein, wherein the chimeric VEGF-binding proteincomprises an immunoglobulin-like domain 2 of a vascular endothelialgrowth factor receptor and an Fc region of immunoglobulin G1 or areduced immunogenic derivative of such an Fc region. Also provided aremethods of using a chimeric VEGF-binding protein comprising animmunoglobulin-like domain 2 of a vascular endothelial growth factorreceptor and an Fc region of immunoglobulin G1 or a reduced immunogenicderivative of such an Fc region in the manufacture for treating anangiogenic disease or disorder in a subject. Also provided are chimericVEGF-binding proteins comprising an immunoglobulin-like domain 2 of avascular endothelial growth factor receptor and an Fc region ofimmunoglobulin G1 or a reduced immunogenic derivative of such an Fcregion for use in treating an angiogenic disease or disorder in asubject.

In another aspect, provided are methods of producing a chimericVEGF-binding protein of the first aspect, the methods comprisingintroducing a recombinant vector encoding such chimeric VEGF-bindingprotein to an isolated host cell, growing or maintaining the cell underconditions permitting the production of the chimeric protein, andrecovering the chimeric protein so produced.

In one embodiment of the treatment methods described herein, a method oftreating cancer is provided, wherein said cancer can be selected, forexample, from the group consisting of glioma, bladder cancer, breastcancer, colon cancer, melanoma, liver cancer, lung cancer, ovariancancer, prostate cancer, renal cell carcinoma, hemangioma andastrocytoma.

In another embodiment of the treatment methods described herein, methodsof treating age-related macular degeneration, wet macular degeneration,and diabetic retinopathy are provided.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Construction of a modified VEGF-Trap protein for gene therapyapplications. Vegf-Trap 1 (VT1) was constructed as described by Holashet al. (1). Vegf-Trap 3 (VT3) was similarly constructed by attaching thesecond Ig-like domain of Flt-1 to Fc-huIgG₁. The final construct encodesthe signal sequence of VEGF-receptor 1 (Amino acids 1-26 of Genbankaccession # BCO39007) (SEQ. ID.No.1), the second Ig-like domain (Aminoacids 129-231 of Genbank accession # BCO39007) (SEQ. ID.No.1) fuseddirectly to the huIgG1 (amino acids 247-473 of Genbank accession #BC092518).

FIG. 2. Deglycosylation of VEGF-Trap 1 and VEGF-Trap 3 using PNGase F,α-2(3,6,8,9)Neuraminidase and O-Glycosidase enzymes. SDS-PAGE (12% gel)followed by Coomassie staining Lane 1 and 4 CandyCane GlycoproteinMolecular weight Marker (Molecular Probes). Lane 2 VEGF-Trap 3. Lane 3VEGF-Trap 3 after deglycosylation. Lane 5 VEGF-Trap 1. Lane 6 VEGF-Trap1 after deglycosylation.

FIG. 3. Concentrations of VEGF-Trap 1 or VEGF-Trap 3 following infectionof 1.7E6 293T cells in culture. Cells were exposed to AAV encodingeither VEGF-Trap 1 or VEGF-Trap 3 at the indicated multiplicity ofinfection.

FIG. 4. Binding affinity analysis of VEGF-binding proteins. In brief, afixed concentration of human VEGF₁₆₅ (10 pM) was incubated overnight atroom temperature with varying concentrations (0.05 pM to 1000 pM) ofVEGF binding proteins. 20 hours later, concentration of unbound VEGF₁₆₅was measured by ELISA. Binding curves were fitted using Prizm software.

FIG. 5. In Vivo pharmacokinetic analysis after subcutaneous injection of100 μg of either VEGF-Trap-1 or VEGF-Trap-3 into Balb/C mice. Mice werebled at 1, 4, 6, 24, 48, 72, 120 hours after injection. Levels ofvarious VEGF binding proteins were measured by ELISA using purifiedVEGF-Trap proteins as standard.

FIG. 6. Plasma concentration of VEGF binding proteins after AAVinjection. Nu/Nu mice were intramuscularly injected with recombinant AAVvectors encoding for either VT1 or VT3. Mice were bled on 4, 7, 8, 1013, 15, 21, 23, 32, 66, 82, 93, 124 days after injection. Plasmaconcentration of VT1 and VT3 was measured by ELISA using human VEGF₁₆₅to bind VEGF-Trap and antibody against human IgG₁-Fc to detect thecaptured proteins.

FIG. 7. Treatment of LLC with VEGF-Trap3 protein. LLC cells wereimplanted subcutaneously in the dorsa of C57B136 mice (n=8). Mice weretreated systemically with 0.025 mg, 0.1 mg or 0.5 mg of VT3 s.c.injections twice week. Control mice were treated with PBS. Tumor volumewas measured every 2 to 4 days. T/C is indicated. VT1 was not detectableup to day 23.

FIG. 8. CD31 staining of LLC tumors treated with three different levelsof VEGF-Trap 3 dosing compared to PBS treated controls. A dose dependentreduction in the number of vessels is observed with increasing VEGF-Trap3 levels.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “chimeric VEGF-binding protein” refers to aprotein comprising a first portion, the first portion consisting ofsections of the Ig-like domains the tyrosine kinase receptor Flt-1, anda second portion comprising a Fc region of immunoglobulin G1 or areduced immunogenic derivative of a Fc region of immunoglobulin G1. Theterms “chimeric VEGF-binding protein”, “VEGF-binding protein” and “VEGFTrap” protein are used interchangeably here. A chimeric VEGF-bindingprotein of this type can specifically bind VEGF as measured in an assayas described herein.

As used herein, the term “chimeric” describes being composed of parts ofdifferent protein or DNA from different origins. For example, a chimericVEGF-binding protein is composed of the Ig-like domain 2 from the Flt-1protein and the Fc region of an immunoglobulin protein. Similarly achimeric polynucleotide is composed of a DNA fragment encoding theIg-like domain 2 of the Flt-1 protein and a DNA fragment encoding the Fcregion of an immunoglobulin protein.

As used herein, the term “Fc region” refers to the fragmentcrystallizable region (Fc region) of an antibody that contributes theconstant domains of an immunoglobulin.

As used herein, the term “reduced immunogenic derivative of an Fcregion” refers to an Fc region that contains certain point mutationsthat render the Fc region less likely to activate and elicit an immuneresponse to the altered Fc region by reducing the ability to bind withthe FcRγ1 receptor on B and T cells. A “reduced immunogenic derivativeof an Fc region” triggers less than 70% of the immune system response ofa wild-type Fc region, and preferably less than 50%, 40%, 20%, 10% orlower relative to wild-type Fc.

The term “vector”, as used herein, refers to a nucleic acid constructdesigned for delivery to a host cell or transfer between different hostcells. As used herein, a vector may be viral or non-viral.

As used herein, the term “expression vector” refers to a vector that hasthe ability to incorporate and express heterologous DNA fragments in acell. An expression vector may comprise additional elements, forexample, the expression vector may have two replication systems, thusallowing it to be maintained in two organisms, for example in humancells for expression and in a prokaryotic host for cloning andamplification.

As used herein, the term “viral vector” refers to a nucleic acid vectorconstruct that includes at least one element of viral origin and has thecapacity to be packaged into a viral vector particle. The viral vectorcan contain the coding sequence for a VEGF-binding protein in place ofnon-essential viral genes. The vector and/or particle may be utilizedfor the purpose of transferring DNA, RNA or other nucleic acids intocells either in vitro or in vivo. Numerous forms of viral vectors areknown in the art.

The term “replication incompetent” as used herein means the viral vectorcannot further replicate and package its genomes. For example, when thecells of a subject are infected with replication incompetent recombinantadeno-associated virus (rAAV) virions, the heterologous (also known astransgene) gene is expressed in the patient's cells, but, the rAAV isreplication defective (e.g., lacks accessory genes that encode essentialproteins from packaging the virus) and viral particles cannot be formedin the patient's cells.

The term “gene” or “coding sequence” means the nucleic acid sequencewhich is transcribed (DNA) and translated (mRNA) into a polypeptide invitro or in vivo when operably linked to appropriate regulatorysequences. The gene may or may not include regions preceding andfollowing the coding region, e.g. 5′ untranslated (5 ‘UTR) or “leader”sequences and 3’ UTR or “trailer” sequences, as well as interveningsequences (introns) between individual coding segments (exons).

The term “recombinant” as used herein with reference to nucleic acidmolecules refers to a combination of nucleic acid molecules that arejoined together using recombinant DNA technology into a progeny nucleicacid molecule. As used herein with reference to viruses, cells, andorganisms, the terms “recombinant,” “transformed,” and “transgenic”refer to a host virus, cell, or organism into which a heterologousnucleic acid molecule has been introduced. The nucleic acid molecule canbe stably integrated into the genome of the host or the nucleic acidmolecule can also be present as an extrachromosomal molecule. Such anextrachromosomal molecule can be auto-replicating. Recombinant viruses,cells, and organisms are understood to encompass not only the endproduct of a transformation process, but also recombinant progenythereof. A “non-transformed,” “non-transgenic,” or “non-recombinant”host refers to a wildtype virus, cell, or organism that does not containthe heterologous nucleic acid molecule.

The term “angiogenesis”, as used herein refers to the sprouting of bloodvessels from pre-existing blood vessels, characterized by endothelialcell proliferation and the proliferation and migration of tube formingcells. Angiogenesis can be triggered by certain pathological conditions,such as the growth of solid tumors and metastasis.

As used herein, the term “angiogenic disease or disorder” refers todiseases or disorders that are the direct result of aberrant bloodvessel proliferation (e.g. diabetic retinopathy). The term also refer todisease or disorder whose pathological progression is dependent on agood blood supply and thus blood vessel proliferation. The term“angiogenesis-related disease or disorder” and “angiogenic disease ordisorder” are used interchangeably herein.

As used herein, the term “variant” refers to the chimeric VEGF-bindingprotein with one or more amino acid changes to the amino acid sequenceof the polypeptide that retains both VEGF-binding activity andanti-angiogenic activity. Thus the polypeptide sequence of a variantchimeric VEGF-binding polypeptide varies from that of, e.g., a constructof SEQ. ID. No. 3.

Chimeric VEGF-Binding Protein

Embodiments of the invention are directed to chimeric VEGF-bindingproteins. Chimeric VEGF-binding proteins as described herein arecomprised of two portions: the first portion consisting of amino acids129-231 of the Flt-1 tyrosine kinase receptor (Genbank Accession No.BCO39007) (SEQ ID NO:1), and a second portion comprising a Fc region ofhuman immunoglobulin G1. In non-limiting embodiments, the second portioncomprising the Fc region can include amino acids 247-473 of IgG1(Genbank Accession No. BC092518) or amino acids 243-469 of SEQ ID NO: 2.

The amino acid sequence of one such chimeric VEGF-binding protein, alsoreferred to herein as VEGF-Trap 3 or VT3, is:

(SEQ. ID. NO. 3) MVSYWDTGVLLCALLSCLLLTGSSSGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

The extracellular regions of the VEGF tyrosine kinase receptors Flt-1and KDR are responsible for binding the ligand VEGF. The extracellularregion has seven immunoglobulin-like (Ig-like) domains. The ligandbinding function resides within the first three domains (Davis-Smyth T.et. al., 1996, J EMBO 15: 4919-4927; Barleon et. al., 1997, J. Biol.Chem. 272:10382-88; Cunningham et. al., 1997, Biochem. Biophys. Res.Comm. 231: 596-599; Wiesmann C. et. al., 1997, Cell 91:695-704). Ofthese seven Ig-like domains, it has been shown that the Ig-like domain 2is essential for binding ligand VEGF, although the Ig-like domain 2(amino acids 134-226) alone was not sufficient for VEGF binding in vitro(Davis-Smyth T. et. al., 1996, J EMBO 15: 4919-4927). As describedherein, surprisingly, a region of the Ig-like domain 2 of Flt-1(aminoacids 129-231) is essential and sufficient for binding VEGF ligand invitro. This Ig-like domain 2 functions effectively in suppressing tumorgrowth in vivo. Since the two high affinity receptor tyrosine kinases,Flt-1 and KDR, share a high degree of sequence similarity and identity,the Ig-like domains 2 of Flt-1 and KDR are interchangeable. Therefore,in one embodiment, the invention encompasses a chimeric VEGF-bindingprotein comprising the Ig-like domain 2 of KDR (amino acids 122-286 ofKDR, genbank Accesion No.: NM002253, SEQ. ID. No. 6).

In one embodiment, a chimeric VEGF-binding protein comprises a firstportion, the first portion consisting of amino acids 129-231 of theFlt-1 tyrosine kinase receptor (SEQ ID No.: 1), and a second portioncomprising an Fc region of immunoglobulin G1 or a reduced immunogenicderivative of an Fc region of immunoglobulin G1. The Fc portion of animmunoglobulin has a long plasma half-life (Capon, et. al., 1989, Nature337: 525-531). The fusion of the single Ig-like domain 2 from eitherFlt-1 or KDR to the Fc portion of an immunoglobulin will serve toimprove the plasma half-life and pharmacokinetics of the VEGF-bindingIgG domain 2 in vivo.

Previously Fc-chimeric proteins have been to shown to elicit aninflammatory response upon long term usage or at high dosage, andespecially when the Fc-chimera is used therapeutically and the Fc regionis derived from a heterologous source different from the animalrecipient of the Fc-chimeric protein. In one embodiment, the Fc regionof immunoglobulin used in the chimeric VEGF-binding protein is a reducedimmunogenic derivative of a Fc region of IgG and is derived from ahuman. The reduced immunogenic activity results from a reduced affinityfor the Fc receptors on B and T cells. The Fc regions of immunoglobulinG subtype 4 (IgG4) and IgG2 have 10-fold lower affinity and no affinityfor the FcRγ1 receptor respectively. In one embodiment, the Fc region ofIgG4 and IgG2 is used in the construction of chimeric VEGF-bindingprotein. In another embodiment, the Fc region has certain pointmutations which further reduces its affinity to the FcRγ1 receptor orthat enhances the serum half-life of the Fc-fusion molecule. Forexample, a serine to proline mutation in residue 241 (kabat numbering)in IgG4 results in increased serum half-life of IgG4 (Angal S., et al.,1993, Mol. Immuno. 130:105-108). A substitution of leucine for glutamateat residue 248 (kabat numbering) in IgG2b decreases the affinity for theFcRγ1 receptor (Canfield and Morrison, J Exp Med. 1991 Jun. 1;173(6):1483-91).

In another embodiment, the chimeric VEGF-binding protein has amino acidpoint mutations and/or substitutions within the Ig-like domain 2 thatenhance the binding affinity of the chimeric VEGF-binding protein forthe homodimeric VEGF ligand. Examples of such mutations include asubstitution of Glu 201 with Asp, Leu 205 with tyrosine, tryptophanreplacing Leu 169 on strand βc (Wiesmann C. et. al., 1997, Cell91:695-704), and replacing Tyr-220 and Arg-224 with hydrophobic aminoacids such as isoleucine and phenylalanine. In one embodiment, variantforms of chimeric VEGF-binding protein may have more than one mutation.In another embodiment, random or systematic mutagenesis can be performedto create a library of variant forms of VEGF-binding protein to screenfor variant forms with greater VEGF-binding. In vitro binding assayswhich can be performed routinely to determine if a particular mutationin the Ig-like domain 2 affects VEGF ligand binding ability aredescribed by Park el. al. (J. Biol. Chem. 1994, 269:25646-54) and HolashJ. et al. (Proc. Natl. Acad. Sci. USA 2002, 99:11393-98) which areincorporated by reference herein. A greater binding affinity as the termis used herein refers to a 5% or greater decrease in K_(d) relative towild-type binding to VEGF.

In one embodiment, a chimeric VEGF-binding protein comprises a signalpeptide at the N-terminus. This signal peptide is a short (3-60 aminoacids long) peptide chain that directs the post-translational transportof a protein. For the current invention, the signal peptide should guidethe DNA sequence encoding the chimeric VEGF-binding protein forco-translation in the endoplasmic reticulum and subsequent secretion ofthe translated protein. In one embodiment, the signal peptide is thesignal peptideH₂N-Met-Met-Ser-Phe-Val-Ser-Leu-Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp-Ala-Thr-Glu-Ala-Glu-Gln-Leu-Thr-Lys-Cys-Glu-Val-Phe-Gln(SEQ. ID. No.: 4). In one embodiment, the chimeric VEGF-binding proteincomprises a signal peptide of the Flt-1 tyrosine kinase receptor (SEQ.ID. No.: 1), amino acids 1-26,Met-Val-Ser-Tyr-Trp-Asp-Thr-Gly-Val-Leu-Leu-Cys-Ala-Leu-Leu-Ser-Cys-Leu-Leu-Leu-Thr-Gly-Ser-Ser-Ser-Gly-Ser(SEQ. ID. No. 5). In another embodiment, the signal peptide is that ofthe KDR tyrosine kinase receptor (Genbank Accession No. NM 002253) (SEQ.ID. No. 6), amino acids 1-26,Met-Gln-Ser-Lys-Val-Leu-Leu-Ala-Val-Ala-Leu-Trp-Leu-Cys-Val-Glu-Thr-Arg-Ala-Ala-Ser-Val-Gly-Leu-Pro-Ser(SEQ. ID. No. 7). In a preferred example, a chimeric VEGF-bindingprotein, as described herein, can have the following protein fragmentsarranged in the amino to carboxyl terminus orientation in thepolypeptide: the signal peptide (amino acids 1-26) the followed by theIg-like domain 2 (amino acids 129-231) of Flt-1 tyrosine kinasereceptor, and ending with the Fc region (amino acids 247-473) of humanIgG1.

Synthesis of Isolated Chimeric DNA Coding Sequence for a ChimericVEGF-Binding Protein

Unless otherwise indicated, all technical and scientific terms usedherein have the same meaning as they would to one skilled in the art ofthe present invention. Further, unless otherwise required by context,singular terms shall include pluralities and plural terms shall includethe singular.

The process of engineering the chimeric protein, the coding DNAsequence, expression vectors, viral vectors, and expression purificationof the invention can be performed by conventional recombinant molecularbiology and protein biochemistry techniques such as those described inManiatis et al. (Molecular Cloning—A Laboratory Manual; Cold SpringHarbor, 1982) and DNA Cloning Vols I, II, and III (D. Glover ed., IRLPress Ltd.), Sambrook et al., (1989, Molecular Cloning, A LaboratoryManual; Cold Spring Harbor Laboratory Press, NY, USA), Current Protocolsin Molecular Biology (CPMB) (Fred M. Ausubel et al. ed., John Wiley andSons, Inc.) and Current Protocols in Protein Science (CPPS) (John E.Coligan, et al., ed., John Wiley and Sons, Inc.).

The term “isolated” refers to material, such as a nucleic acid or aprotein, which is: (1) substantially or essentially free from componentswhich normally accompany or interact with the material as found in itsnaturally occurring environment or (2) if the material is in its naturalenvironment, the material has been altered by deliberate humanintervention to a composition and/or placed at a locus in the cell otherthan the locus native to the material.

Conventional polymerase chain reaction (PCR) cloning techniques can beused to generate an isolated chimeric DNA sequence encoding the chimericVEGF-binding protein. A chimeric DNA sequence is cloned into a generalpurpose cloning vector such as pUC19, pBR322, pBluescript vectors(Stratagene Inc.) or pCR TOPO® from Invitrogen Inc. The resultantrecombinant vector carrying the isolated chimeric DNA sequence encodinga chimeric VEGF-binding protein can then be used for further molecularbiological manipulations such as site-directed mutagenesis to enhanceVEGF-binding and/or to reduce the immunogenic properties of the chimericprotein, or can be subcloned into protein expression vectors or viralvectors for protein synthesis in a variety of protein expression systemsusing host cells selected from the group consisting of mammalian celllines, insect cell lines, yeast, bacteria, and plant cells.

Embodied herein is an isolated polynucleotide encoding a chimericVEGF-binding protein comprising a first portion, the first portionconsisting of amino acids 129-231 of the Flt-1 tyrosine kinase receptor(SEQ ID No.: 1), and a second portion comprising a Fc region ofimmunoglobulin G1 or a reduced immunogenic derivative of a Fc region ofimmunoglobulin G1. The single isolated DNA encoding the chimericVEGF-binding protein is made three up of separate DNA fragments, eachfragment coding for an individual part of the chimeric protein: thesignal peptide, the Ig-like domain 2 of Flt-1, and the Fc region of anIgG. Three pairs of specific PCR oligonucleotide primers can be used toPCR amplify the three separate DNA fragments corresponding to the aminoacids 1-26 and 129-231 of the Flt-1 tyrosine kinase receptor (Genbankaccession # BCO39007) (SEQ ID No.: 1), and the amino acids 247-473 ofhuman IgG1 (Genbank accession # BC092518). Each PCR primer should haveat least 15 nucleotides overlapping with its corresponding templates atthe region to be amplified. The polymerase used in the PCR amplificationshould have high fidelity such as Strategene's PfuUltra™ polymerase forreducing sequence mistakes during the PCR amplification process. Forease of ligating the three separate PCR fragments together and theninserting into a cloning vector, the PCR primers should also havedistinct and unique restriction digestion sites on their flanking endsthat do not anneal to the DNA template during PCR amplification. Thechoice of the restriction digestion sites for each pair of specificprimers should be such that the open reading frame of the chimeric DNAsequence is in-frame and will encode the predicted chimeric VEGF-bindingprotein from beginning to end with no stop codons. At the same time thechosen restriction digestion sites should not be found within the DNAcoding sequences corresponding to the amino acids 1-26 and 129-231 ofthe Flt-1 tyrosine kinase receptor (SEQ. ID. No.:1), and the amino acids247-473 of human IgG1 (Genbank accession # BC092518). Conventionalrestriction digestion and ligation techniques can be used to insert thechimeric DNA coding sequence into a cloning vector. Alternatively thechimeric DNA can be ligated into a vector using the TOPO® cloning methodin Invitrogen topoisomerase-assisted TA vectors such as pCR®-TOPO,pCR®-Blunt II-TOPO, pENTR/D-TOPO, and pENTR/SD/D-TOPO®. BothpENTR/D-TOPO®, and pENTR/SD/D-TOPO® are directional TOPO entry vectorswhich allow the cloning of the chimeric VEGF DNA sequence in the 5′→3′orientation into a Gateway® expression vector. Directional cloning inthe 5′→3′ orientation facilitate the unidirection insertion of thechimeric VEGF DNA sequence into a protein expression vector such thatthe promoter is upstream of the 5′ ATG start codon of the chimeric VEGFDNA sequence, enabling promoter driven protein expression. Therecombinant vector carrying the chimeric VEGF DNA sequence can betransfected into and propagated in general cloning E. coli such asXL1Blue, SURE (Stratagene) and TOP-10 cells (Invitrogen).

In one embodiment, the invention provides an isolated polynucleotideencoding a chimeric VEGF-binding protein. An example of such an isolatedpolynucleotide is:

(SEQ. ID. No. 8) Atggtgagctactgggacactggggtgctgctgtgtgccctgctgagctgcctgctgctgactggcagcagctctggctctgacactggcaggccctttgtggagatgtactctgagatccctgagatcatccacatgactgagggcagggagctggtgatcccctgcagagtgaccagccccaacatcactgtgaccctgaagaagttccccctggacaccctgatccctgatggcaagaggatcatctgggacagcaggaagggcttcatcatcagcaatgccacctacaaggagattggcctgctgacctgtgaggccactgtgaatggccacctgtacaagaccaactacctgacccacaggcagaccaacaccatcatcgatgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga-3′.

Specific site-directed mutagenesis of the chimeric VEGF DNA sequence ina vector can be used to create specific amino acid mutations andsubstitutions at the Fc portion to further reduce the immunogenicproperties of eventual chimeric VEGF-binding protein. By the same token,site-directed mutagenesis can be carried out in the Ig-like domain 2 toenhance the VEGF-binding affinity of the chimeric VEGF-binding protein.Examples of amino acid mutations are serine to proline mutation and asubstitution of leucine for glutamate in the Fc-region. Site-directedmutagenesis may be carried out using the QuikChange® site-directedmutagenesis kit from Stratagene according to manufacture's instructionsor any methods known in the art.

Expression Vectors and Expression Systems for Expression of ChimericVEGF-Binding Protein

In one embodiment, the invention provides for expression vectorscarrying a polynucleotide that encodes a chimeric VEGF-binding proteinfor the expression and purification of the recombinant chimericVEGF-binding protein produced from a protein expression system usinghost cells selected from, e.g., mammalian, insect, yeast, bacterial, orplant cells.

In one embodiment, the recombinant vector that expresses a chimericVEGF-binding protein is a viral vector. The viral vector can be anyviral vector known in the art including but not limited to those derivedfrom adenovirus, adeno-associated virus (AAV), retrovirus, andlentivirus. Recombinant viruses provide a versatile system for geneexpression studies and therapeutic applications.

In another embodiment, the invention provides for a host cell comprisinga expression vector which expresses the chimeric VEGF-binding protein.The expression host cell may be derived from any of a number of sources,e.g., bacteria, such as E. coli, yeasts, mammals, insects, and plantcells such as chymadomonas. In another embodiment, the recombinantchimeric VEGF-binding protein can be produced from expression vectorssuitable for cell-free expression systems. From the cloning vector, thechimeric VEGF DNA sequence can be subcloned into a recombinantexpression vector that is appropriate for the expression of the chimericVEGF-binding protein in mammalian, insect, yeast, bacterial, or plantcells or a cell-free expression system such as a rabbit reticulocyteexpression system. Subcloning can be achieved by PCR cloning,restriction digestion followed by ligation, or recombination reactionsuch as those of the lambda phage-based site-specific recombinationusing the Gateway® LR and BP Clonase™ enzyme mixtures. Subcloning shouldbe unidirectional such that the 5′ ATG start codon of the chimeric VEGFDNA sequence is downstream of the promoter in the expression vector.Alternatively, when the chimeric VEGF DNA sequence is cloned intopENTR/D-TOPO®, pENTR/SD/D-TOPO® (directional entry vectors), or any ofthe Invitrogen's Gateway® Technology pENTR (entry) vectors, the chimericVEGF DNA sequence can be transferred into the various Gateway®expression vectors (destination) for protein expression in mammaliancells, E. coli, insects and yeast respectively in one singlerecombination reaction. Some of the Gateway® destination vectors aredesigned for the constructions of baculovirus, adenovirus,adeno-associated virus (AAV), retrovirus, and lentiviruses, which uponinfecting their respective host cells, permit heterologous expression ofthe chimeric VEGF-binding protein in the host cells. The Gateway®Technology uses lambda phage-based site-specific recombination insteadof restriction endonuclease and ligase to insert a gene of interest intoan expression vector. The DNA recombination sequences (attL, attR, attB,and attP) and the LR and BP Clonase™ enzyme mixtures that mediate thelambda recombination reactions are the foundation of Gateway®Technology. Transferring a gene into a destination vector isaccomplished in just two steps: Step 1: Clone the chimeric DNA sequenceinto an entry vector such as pENTR/D-TOPO®. Step 2: Mix the entry clonecontaining the chimeric DNA sequence in vitro with the appropriateGateway® expression vector (destination vector) and Gateway® LR Clonase™enzyme mix. There are Gateway® expression vectors for protein expressionin E. coli, insect cells, mammalian cells, and yeast. Site-specificrecombination between the att sites (attR×attL and attB×attP) generatesan expression vector and a by-product. The expression vector containsthe chimeric DNA sequence recombined into the destination vectorbackbone. Following transformation and selection in E. coli, theexpression vector is ready to be used for expression in the appropriatehost.

The expression vector should have the necessary 5′ upstream and 3′downstream regulatory elements such as promoter sequences, ribosomerecognition and binding TATA box, and 3′ UTR AAUAAA transcriptiontermination sequence for the efficient gene transcription andtranslation in its respective host cell. The expression vector may haveadditional sequence such as 6X-histidine, V5, thioredoxin,glutathione-S-transferase, c-Myc, VSV-G, HSV, FLAG, maltose bindingpeptide, metal-binding peptide, HA and “secretion” signals (Honeybeemelittin, α-factor, PHO, Bip), which are incorporated into the expressedrecombinant chimeric VEGF-binding protein. In addition, there may beenzyme digestion sites incorporated after these sequences to facilitateenzymatic removal of them after they are not needed. These additionalsequences are useful for the detection of the chimeric VEGF-bindingprotein expression, for protein purification by affinity chromatography,enhanced solubility of the recombinant protein in the host cytoplasm,and/or for secreting the expressed recombinant chimeric VEGF-bindingprotein out into the culture media, into the periplasm of the prokaryotebacteria, or the spheroplast of the yeast cells. The expression of therecombinant chimeric VEGF-binding protein can be constitutive in thehost cells or it can be induced, e.g., with copper sulfate, sugars suchas galactose, methanol, methylamine, thiamine, tetracycline, infectionwith baculovirus, and (isopropyl-beta-D-thiogalactopyranoside) IPTG, astable synthetic analog of lactose.

Examples of expression vectors and host cells are the pET vectors(Novagen), pGEX vectors (Amersham Pharmacia), and pMAL vectors (NewEngland labs. Inc.) for protein expression in E. coli host cell such asBL21, BL21(DE3) and AD494(DE3)pLysS, Rosetta (DE3), and Origami(DE3)(Novagen); the strong CMV promoter-based pcDNA3.1 (Invitrogen) andpCIneo vectors (Promega) for expression in mammalian cell lines such asCHO, COS, HEK-293, Jurkat, and MCF-7; replication incompetent adenoviralvector vectors pAdeno X, pAd5F35, pLP-Adeno-X-CMV (Clontech),pAd/CMVN5-DEST, pAd-DEST vector (Invitrogen) for adenovirus-mediatedgene transfer and expression in mammalian cells; pLNCX2, pLXSN, andpLAPSN retrovirus vectors for use with the Retro-X™ system from Clontechfor retroviral-mediated gene transfer and expression in mammalian cells;pLenti4/V5-DEST™, pLenti6/V5-DEST™, and pLenti6.2/V5-GW/lacZ(Invitrogen) for lentivirus-mediated gene transfer and expression inmammalian cells; adenovirus-associated virus expression vectors such aspAAV-MCS, pAAV-IRES-hrGFP, and pAAV-RC vector (Stratagene) foradeno-associated virus-mediated gene transfer and expression inmammalian cells; BACpak6 baculovirus (Clontech) and pFastBac™ HT(Invitrogen) for the expression in Spodopera frugiperda 9 (Sf9) and Sf11insect cell lines; pMT/BiP/V5-His (Invitrogen) for the expression inDrosophila Schneider S2 cells; Pichia expression vectors pPICZα, pPICZ,pFLDα and pFLD (Invitrogen) for expression in Pichia pastoris andvectors pMETα and pMET for expression in P. methanolica; pYES2/GS andpYD1 (Invitrogen) vectors for expression in yeast Saccharomycescerevisiae. Recent advances in the large scale expression heterologousproteins in Chlamydomonas reinhardtii are described by Griesbeck C. et.al. 2006 Mol. Biotechnol. 34:213-33 and Fuhrmann M. 2004, Methods MolMed. 94:191-5. Foreign heterologous coding sequences are inserted intothe genome of the nucleus, chloroplast and mitochodria by homologousrecombination. The chloroplast expression vector p64 carrying the mostversatile chloroplast selectable marker aminoglycoside adenyltransferase (aadA), which confer resistance to spectinomycin orstreptomycin, can be used to express foreign protein in the chloroplast.Biolistic gene gun method is used to introduced the vector in the algae.Upon its entry into chloroplasts, the foreign DNA is released from thegene gun particles and integrates into the chloroplast genome throughhomologous recombination.

A simplified system for generating recombinant adenoviruses is presentedby He TC. et. al. Proc. Natl. Acad. Sci. USA 95:2509-2514, 1998. Thegene of interest is first cloned into a shuttle vector, e.g.pAdTrack-CMV. The resultant plasmid is linearized by digesting withrestriction endonuclease Pme I, and subsequently cotransformed into E.coli. BJ5183 cells with an adenoviral backbone plasmid, e.g. pAdEasy-1of Stratagene's AdEasy™ Adenoviral Vector System. Recombinant adenovirusvectors are selected for kanamycin resistance, and recombinationconfirmed by restriction endonuclease analyses. Finally, the linearizedrecombinant plasmid is transfected into adenovirus packaging cell lines,for example HEK 293 cells(E1-transformed human embryonic kidney cells)or 911 (E1-transformed human embryonic retinal cells) (Human GeneTherapy 7:215-222, 1996). Recombinant adenovirus are generated withinthe HEK 293 cells.

In one embodiment, the invention provides a recombinant lentivirus forthe delivery and expression of a chimeric VEGF-binding protein in eitherdividing and non-dividing mammalian cells. The HIV-1 based lentiviruscan effectively transduce a broader host range than the Moloney LeukemiaVirus (MoMLV)-base retroviral systems. Preparation of the recombinantlentivirus can be achieved using the pLenti4/V5-DEST™, pLenti6/V5-DEST™or pLenti vectors together with ViraPower™ Lentiviral Expression systemsfrom Invitrogen.

In one embodiment, the invention provides a recombinant adeno-associatedvirus (rAAV) vector for the expression of a chimeric VEGF-bindingprotein. In one embodiment, the rAAV vector encoding a chimericVEGF-binding protein is administered to slow, inhibit, or prevent thegrowth of cancer and tumors such as glioma. Using rAAV vectors, genescan be delivered into a wide range of host cells including manydifferent human and non-human cell lines or tissues. Because AAV isnon-pathogenic and does not illicit an immune response, a multitude ofpre-clinical studies have reported excellent safety profiles. rAAVs arecapable of transducing a broad range of cell types and transduction isnot dependent on active host cell division. High titers, >10⁸ viralparticle/ml, are easily obtained in the supernatant and 10¹¹-10¹² viralparticle/ml with further concentration. The transgene is integrated intothe host genome so expression is long term and stable.

The use of alternative AAV serotypes other than AAV-2 (Davidson et al(2000), PNAS 97(7)3428-32; Passini et al (2003), J. Virol77(12):7034-40) has demonstrated different cell tropisms and increasedtransduction capabilities. With respect to brain cancers, thedevelopment of novel injection techniques into the brain, specificallyconvection enhanced delivery (CED; Bobo et al (1994), PNAS91(6):2076-80; Nguyen et al (2001), Neuroreport 12(9):1961-4), hassignificantly enhanced the ability to transduce large areas of the brainwith an AAV vector.

Large scale preparation of AAV vectors is made by a three-plasmidcotransfection of a packaging cell line: AAV vector carrying thechimeric DNA coding sequence, AAV RC vector containing AAV rep and capgenes, and adenovirus helper plasmid pDF6, into 50×150 mm plates ofsubconfluent 293 cells. Cells are harvested three days aftertransfection, and viruses are released by three freeze-thaw cycles or bysonication.

AAV vectors are then purified by two different methods depending on theserotype of the vector. AAV2 vector is purified by the single-stepgravity-flow column purification method based on its affinity forheparin (Auricchio, A., et. al., 2001, Human Gene therapy 12; 71-6;Summerford, C. and R. Samulski, 1998, J. Virol. 72:1438-45; Summerford,C. and R. Samulski, 1999, Nat. Med. 5: 587-88). AAV2/1 and AAV2/5vectors are currently purified by three sequential CsCl gradients.

Expression and Purification

In one embodiment, the invention provides a method of producing chimericVEGF-binding protein comprising introducing the recombinant vector thatexpressed the chimeric VEGF-binding protein into an isolated host cell,growing the cell under conditions permitting the production of thechimeric protein and recovering the chimeric VEGF-binding protein soproduced. The methods described herein provide for the expression andpurification of the chimeric VEGF protein in various cell-basedexpression systems such as protein production in bacterial, mammalian,insect, yeast, and chymadomonas cells. Protein expression can beconstitutive or inducible with inducers such as copper sulfate, sugarssuch as galactose, methanol, methylamine, thiamine, tetracycline, orIPTG. After the protein is expressed in the host cells, the host cellsare lysed to liberate the expressed protein for purification. Methods oflysing the various host cells are featured in “Sample Preparation-Toolsfor Protein Research” EMD Bioscience and in the Current Protocols inProtein Sciences (CPPS). The preferred purification method is affinitychromatography such as ion-metal affinity chromatograph using nickel,cobalt, or zinc affinity resins for histidine-tagged chimericVEGF-binding protein. Methods of purifying histidine-tagged recombinantproteins are described by Clontech using their Talon® cobalt resin andby Novagen in their pET system manual, 10^(th) edition. Anotherpreferred purification strategy is by immuno-affinity chromatography,for example, anti-myc antibody conjugated resin can be used to affinitypurify myc-tagged chimeric VEGF-binding protein. Enzymatic digestionwith serine proteases such as thrombin and enterokinase cleave andrelease the chimeric VEGF-binding protein from the histidine or myc tag,releasing the chimeric VEGF-binding protein from the affinity resinwhile the histidine-tags and myc-tags are left attached to the affinityresin.

Besides cell-based expression systems, cell-free expression systems arealso contemplated. Cell-free expression systems offer several advantagesover traditional cell-based expression methods, including the easymodification of reaction conditions to favor protein folding, decreasedsensitivity to product toxicity and suitability for high-throughputstrategies such as rapid expression screening or large amount proteinproduction because of reduced reaction volumes and process time. Thecell-free expression system can use plasmid or linear DNA. Moreover,improvements in translation efficiency have resulted in yields thatexceed a milligram of protein per milliliter of reaction mix.

In one embodiment, a continuous cell-free translation system may be usedto produce a chimeric VEGF-binding protein. A continuous cell-freetranslation system capable of producing proteins in high yield isdescribed by Spirin A S. et. al., Science 242:1162 (1988). The methoduses a continuous flow design of the feeding buffer which contains aminoacids, adenosine triphosphate (ATP), and guanosine triphosphate (GTP)throughout the reaction mixture and a continuous removal of thetranslated polypeptide product. The system uses E. coli lysate toprovide the cell-free continuous feeding buffer. This continuous flowsystem is compatible with both prokaryotic and eukaryotic expressionvectors. Large scale cell-free production of the integral membraneprotein EmrE multidrug transporter is described by Chang G. el. al.,Science 310:1950-3 (2005).

Other commercially available cell-free expression systems include theExpressway™ Cell-Free Expression Systems (Invitrogen) which utilize anE. coli-based in-vitro system for efficient, coupled transcription andtranslation reactions to produce up to milligram quantities of activerecombinant protein in a tube reaction format; the Rapid TranslationSystem (RTS) (Roche Applied Science) which also uses an E. coli-basedin-vitro system; and the TNT Coupled Reticulocyte Lysate Systems(Promega) which uses rabbit reticulocyte-based in-vitro system.

Therapeutic Uses and Formulation

In one embodiment, the invention provides a method of treating anangiogenic disease or disorder, comprising administering to a subject inneed thereof a vector comprising a polynucleotide encoding a chimericVEGF binding polypeptide, wherein the chimeric VEGF-binding polypeptidecomprises an Ig-like domain 2 of a vascular endothelial growth factorreceptor and an Fc region of immunoglobulin G1 or a reduced immunogenicderivative of an Fc region of immunoglobulin G1. Alternatively, one canadminister a pharmaceutical composition comprising a chimeric VEGFbinding protein and a pharmaceutically acceptable carrier. In oneembodiment, the said subject in need of treatment can be a mammal, suchas a dog or a cat, preferably a human.

In another embodiment, the invention provides for a method of treatingan angiogenesis-related disease or disorder, comprising administering toa subject in need thereof a pharmaceutical composition comprising achimeric protein, wherein the chimeric polypeptide comprises animmunoglobulin-like domain 2 of a vascular endothelial growth factorreceptor and an Fc region of immunoglobulin G1 or a reduced immunogenicderivative of an Fc region of immunoglobulin G1. In another embodiment,the methods described herein can be used in combination with othertreatment options available for angiogenesis-related diseases ordisorders.

Angiogenesis, as used herein refers to the sprouting of new bloodvessels from pre-existing blood vessels, characterized by endothelialcell proliferation and migration triggered by certain pathologicalconditions, such as the growth of solid tumors and metastasis.

As used herein, the term “angiogenesis-related disease or disorder”refers to diseases or disorders that are dependent on a rich bloodsupply and blood vessel proliferation for the disease's pathologicalprogression (eg. metastatic tumors) or diseases or disorders that arethe direct result of aberrant blood vessel proliferation (e.g. diabeticretinopathy and hemangiomas). Examples include abnormal vascularproliferation, ascites formation, psoriasis, age-related maculardegeneration, thyroid hyperplasia, preclampsia, rheumatoid arthritis andosteo-arthritis, Alzheimer's disease, obesity, pleural effusion,atherosclerosis, endometriosis, diabetic/other retinopathies, ocularneovascularizations such as neovascular glauocoma and cornealneovascularization.

The angiogenesis-related disease or disorder can be selected, forexample, from a group consisting of cancer, ascites formation,psoriasis, age-related macular degeneration, thyroid hyperplasia,preclampsia, rheumatoid arthritis and osteoarthritis, Alzheimer'sdisease, obesity, pleura effusion, atherosclerosis, endometriosis,diabetic/other retinopathies, neovascular glauocoma, age-related maculardegeneration, hemangiomas, and corneal neovascularization. In oneembodiment, the age-related macular degeneration is wet maculardegeneration.

In one embodiment, the anigiogensis-related disease or disorder iscancer, where the rapidly dividing neoplastic cancer cells require anefficient blood supply to sustain their continual growth of the tumor.As used herein, cancer refers to any of various malignant neoplasmscharacterized by the proliferation of anaplastic cells that tend toinvade surrounding tissue and metastasize to new body sites and alsorefers to the pathological condition characterized by such malignantneoplastic growths. The blood vessels provide conduits to metastasizeand spread elsewhere in the body. Upon arrival at the metastatic site,the cancer cells then work on establishing a new blood supply network.Administration of a polynucleotide encoding a chimeric VEGF bindingpolypeptide or a pharmaceutical composition comprising a chimeric VEGFbinding protein and a pharmaceutically acceptable carrier can inhibitangiogenesis. By inhibiting angiogensis at the primary tumor site andsecondary tumor site, embodiments of the invention serve to prevent andlimit the progression of the disease.

The effectiveness of a given VEGF-binding chimeric polypeptide asdescribed herein can be evaluated in vitro or in vivo or both, asdescribed, e.g., in the Examples provided herein below. For theavoidance of doubt, one can also use other assays commonly accepted inthe field. For example, one can use the “CAM” assay. The chickchorioallantoic membrane (CAM) assay is frequently used to evaluate theeffects of angiogenesis regulating factors because it is relatively easyand provides relatively rapid results. A chimeric VEGF-bindingpolypeptide useful in the methods and compositions described herein willdecrease the number of microvessels in the modified CAM assay describedby Iruela-Arispe et al., 1999, Circulation 100: 1423-1431 (incorporatedherein by reference), relative to controls with no chimeric polypeptideadded or expressed. The method is based on the vertical growth of newcapillary vessels into a collagen gel pellet placed on the CAM. In theassay as described by Iruela-Arispe et al., the collagen gel issupplemented with VEGF (250 ng/gel) in the presence or absence of testproteins/peptides. The extent of the anti-angiogenic effect is measuredusing FITC-dextran (50 μg/mL) (Sigma) injected into the circulation ofthe CAM. The degree of fluorescence intensity parallels variations incapillary density; the linearity of this correlation can be observedwith a range of capillaries between 5 and 540. Morphometric analyses areperformed, for example, by acquisition of images with a CCD camera.Images are then analyzed by importing into an analysis package, e.g.,NHlmage 1.59, and measurements of fluorescence intensity are obtained aspositive pixels. Each data point is compared with its own positive andnegative controls present in the same CAM and interpreted as apercentage of inhibition, considering the positive control to be 100%(VEGF alone) and the negative control (vehicle alone) 0%. Statisticalevaluation of the data is performed to check whether groups differsignificantly from random, e.g., by analysis of contingency with Yates'correction.

Additional angiogenesis assays are known in the art and can be used toevaluate chimeric VEGF-binding polypeptides for use in the methods andcompositions described herein. These include, for example, the cornealmicropocket assay, hamster cheek pouch assay, the Matrigel assay andmodifications thereof, and co-culture assays. Donovan et al. describe acomparison of three different in vitro assays developed to evaluateangiogenesis regulators in a human background (Donovan et al., 2001,Angiogenesis 4: 113-121, incorporated herein by reference). Briefly, theassays examined include: 1) a basic Matrigel assay in which low passagehuman endothelial cells (Human umbilical vein endothelial cells, HUVEC)are plated in wells coated with Matrigel (Becton Dickinson, Cedex,France) with or without angiogenesis regulator(s); 2) a similar Matrigelassay using “growth factor reduced” or GFR Matrigel; and 3) a co-cultureassay in which primary human fibroblasts and HUVEC are co-cultured withor without additional angiogenesis regulator(s)—the fibroblasts produceextracellular matrix and other factors that support HUVECdifferentiation and tubule formation. In the Donovan et al. paper theco-culture assay provided microvessel networks that most closelyresembled microvessel networks in vivo. However, the basic Matrigelassay and the GFR Matrigel assay can also be used by one of skill in theart to evaluate whether a given chimeric VEGF-binding polypeptide is anangiogenesis inhibitor as necessary for the methods and compositionsdescribed herein. Finally, an in vitro angiogenesis assay kit ismarketed by Chemicon (Millipore). The Fibrin Gel In Vitro AngiogenesisAssay Kit is Chemicon Catalog No. ECM630. A chimeric VEGF-bindingpolypeptide as described herein is considered useful in a method orcomposition for treatment of an angiogenesis-related disease or disorderas described herein if it reduces angiogenesis in any one of theseassays by 10% or more relative to a control assay performed without thechimeric VEGF-binding polypeptide. A chimeric VEGF-binding polypeptideas described herein preferably reduces angiogenesis in one or more ofthese assays by at least 20%, at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90% or more, up toand including 100% inhibition.

Alternatively, angiogenesis inhibition can be measured functionallydownstream, as a reduction or cessation of tumor growth or tumor size.For example, if there is zero growth of tumor mass, or at least 5%reduction in the size of the tumor mass, there is angiogenesisinhibition by a composition or method as described herein.

Any solid tumor that requires an efficient blood supply to keep growingis a candidate target. For example, candidates for the treatment methodsdescribed herein include carcinomas and sarcomas found in the anus,bladder, bile duct, bone, brain, breast, cervix, colon/rectum,endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney,larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis,prostate, skin, small intestine, stomach, spinal marrow, tailbone,testicles, thyroid and uterus. The types of carcinomas includepapilloma/carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma,adenoma/adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma,rhabdomyoma, mesothelioma, angioma, osteoma, chondroma, glioma,lymphoma/leukemia, squamous cell carcinoma, small cell carcinoma, largecell undifferentiated carcinomas, basal cell carcinoma and sinonasalundifferentiated carcinoma. The types of sarcomas include soft tissuesarcoma such as alveolar soft part sarcoma, angiosarcoma,dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor,extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma,hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma,liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibroushistiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, andAskin's tumor, Ewing's sarcoma (primitive neuroectodermal tumor),malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, andchondrosarcoma. Abnormal build up and growth of blood vessels in theskin or internal organs in the form of hemangiomas can also be treatedaccording to the methods described herein.

In another embodiment, the invention can be used in preventing blindingblood vessel growth associated with diabetic eye diseases, namelydiabetic retinopathy. The methods described herein are designed toantagonize vascular endothelial growth factor (VEGF), a substancenaturally produced in the body that promotes blood vessel formation.Released by the retina (light-sensitive tissue in back of the eye) whennormal blood vessels are damaged by tiny blood clots due to diabetes,VEGF turns on its receptor, igniting a chain reaction that culminates innew blood vessel growth. However, the backup blood vessels are faulty;they leak, bleed and encourage scar tissue that detaches the retina,resulting in severe loss of vision. Such growth is the hallmark ofdiabetic retinopathy, the leading cause of blindness among young peoplein developed countries.

In yet another embodiment, the invention may be used in the treatment ofage-related macular degeneration, as it is known that VEGF alsocontributes to abnormal blood vessel growth from the choroid layer ofthe eye into the retina, similar to what occurs during the wet orneovascular form of age-related macular degeneration. Maculardegeneration, often called AMD or ARMD (age-related maculardegeneration), is the leading cause of vision loss and blindness inAmericans aged 65 and older. New blood vessels grow (neovascularization)beneath the retina and leak blood and fluid. This leakage causespermanent damage to light-sensitive retinal cells, which die off andcreate blind spots in central vision or the macula.

In one embodiment, the angiogenesis-related disease or disorder isrheumatoid arthritis. Rheumatoid arthritis (RA) is characterized bysynovial tissue swelling, leucocyte ingress and angiogenesis, or newblood vessel growth. The disease is thought to occur as an immunologicalresponse to an as yet unidentified antigen. The expansion of thesynovial lining of joints in rheumatoid arthritis (RA) and thesubsequent invasion by the pannus of underlying cartilage and bonenecessitate an increase in the vascular supply to the synovium, to copewith the increased requirement for oxygen and nutrients. Angiogenesis isnow recognised as a key event in the formation and maintenance of thepannus in RA (Paleolog, E. M., 2002, Arthritis Res. 4(Suppl 3):S81-S90).Even in early RA, some of the earliest histological observations areblood vessels. A mononuclear infiltrate characterizes the synovialtissue along with a luxuriant vasculature. Angiogenesis is integral toformation of the inflammatory pannus and without angiogenesis, leukocyteingress could not occur (Koch, A. E., 2000, Ann. Rheum. Dis.; 59(Suppl1):i65-i71). Disruption of the formation of new blood vessels would notonly prevent delivery of nutrients to the inflammatory site, it couldalso reduce joint swelling due to the additional activity of VEGF, apotent pro-angiogenic factor in RA, as a vascular permeability factor.

In one embodiment, the angiogenesis-related disease or disorder isAlzheimer's disease. Alzheimer's disease (AD) is the most common causeof dementia worldwide. AD is characterized by an excessive cerebralamyloid deposition leading to degeneration of neurons and eventually todementia. The exact cause of AD is still unknown. It has been shown byepidemiological studies that long-term use of non-steroidalanti-inflammatory drugs, statins, histamine H2-receptor blockers, orcalcium-channel blockers, all of which are cardiovascular drugs with ananti-angiogenic effects, seem to prevent Alzheimer's disease and/orinfluence the outcome of AD patients. Therefore, it has been speculatedthat in AD angiogenesis in the brain vasculature may play an importantrole in AD. In Alzheimer's disease, the brain endothelium secretes theprecursor substrate for the beta-amyloid plaque and a neurotoxic peptidethat selectively kills cortical neurons. Moreover amyloid deposition inthe vasculature leads to endothelial cell apoptosis and endothelial cellactivation which leads to neovascularization. Vessel formation could beblocked by the VEGF antagonist SU 4312 as well as by statins, indicatingthat anti-angiogenesis strategies based on VEGF inhibition can interferewith endothelial cell activation in AD (Schultheiss C., el. al., 2006,Angiogenesis. 9(2):59-65; Grammas P., et. al., 1999, Am. J. Path.,154(2):337-42) and can be used for preventing and/or treating AD.

In one embodiment, the angiogenesis-related disease or disorder isobesity. It has been shown that the angiogenesis inhibitor, TNP-470 wasable to prevent diet-induced and genetic obesity in mice (EbbaBråkenhielm et. al., Circulation Research, 2004; 94:1579). TNP-470reduced vascularity in the adipose tissue, thereby inhibiting the rateof growth of the adipose tissue and obesity development. Thus,inhibition of angiogenesis can be therapeutic for obesity.

In one embodiment, the angiogenesis-related disease or disorder isendometriosis. Excessive endometrial angiogenesis is proposed as animportant mechanism in the pathogenesis of endometriosis (Healy, D L.,et. al., 1998, Human Reproduction Update, 4:736-740). The endometrium ofpatients with endometriosis shows enhanced endothelial cellproliferation. Moreover there is an elevated expression of the celladhesion molecule integrin v133 in more blood vessels in the endometriumof women with endometriosis when compared with normal women. Strategiesthat inhibit angiogenesis can be used to treat endometriosis.

In one embodiment, the invention provides for a pharmaceuticalcomposition comprising a chimeric VEGF-binding protein and apharmaceutically acceptable carrier. In another embodiment, theinvention provides for a pharmaceutical composition comprising anexpression vector carrying a chimeric DNA sequence that encodes thechimeric VEGF-binding protein and a pharmaceutically acceptable carrier.In yet another embodiment, the invention provides for a pharmaceuticalcomposition comprising the host viral cells (vectors) harboring thechimeric DNA sequence that encodes the chimeric VEGF-binding protein anda pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carrierof chemicals and compounds commonly used in the pharmaceutical industry.The term “pharmaceutically acceptable carries” excludes tissue culturemedium.

For angiogenic diseases or disorders that are accessible externally onthe skin, such as dermal hemangiomas and skin cancer lesions (melanoma),gene therapy virus, expression vectors, or chimeric VEGF-binding proteincan be preferably applied topically to the hemangioma or cancer lesionsite in a therapeutically effective amount in admixture withpharmaceutical carriers, in the form of topical pharmaceuticalcompositions. The gene therapy virus can be in the form of anadenovirus, adeno-associated virus or lentivirus. Such compositionsinclude solutions, suspensions, lotions, gels, creams, ointments,emulsions, skin patches, etc. All of these dosage forms, along withmethods for their preparation, are well known in the pharmaceutical andcosmetic art. HARRY'S COSMETICOLOGY (Chemical Publishing, 7th ed. 1982);REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Co., 18th ed.1990). Typically, such topical formulations contain the activeingredient in a concentration range of 0.1 to 100 mg/ml, in admixturewith suitable vehicles. A suitable vehicle will not promote an immuneresponse to the chimeric polypeptides described herein. For gene therapyviruses, the dosage ranges from 10⁽⁶⁾ to 10⁽¹⁴⁾ particles perapplication. Other desirable ingredients for use in such preparationsinclude preservatives, co-solvents, viscosity building agents, carriers,etc. The carrier itself or a component dissolved in the carrier may havepalliative or therapeutic properties of its own, including moisturizing,cleansing, or anti-inflammatory/anti-itching properties. Penetrationenhancers may, for example, be surface active agents; certain organicsolvents, such as di-methylsulfoxide and other sulfoxides,dimethyl-acetamide and pyrrolidone; certain amides of heterocyclicamines, glycols (e.g. propylene glycol); propylene carbonate; oleicacid; alkyl amines and derivatives; various cationic, anionic, nonionic,and amphoteric surface active agents; and the like.

Topical administration of a pharmacologically effective amount mayutilize transdermal delivery systems well known in the art. An exampleis a dermal patch. Alternatively the biolistic gene gun method ofdelivery may be used. The gene gun is a device for injecting cells withgenetic information, originally designed for plant transformation. Thepayload is an elemental particle of a heavy metal coated with plasmidDNA. This technique is often simply referred to as biolistics. Anotherinstrument that uses biolistics technology is the PDS-1000/He particledelivery system. The chimeric VEGF-binding protein, expression vector,and/or gene therapy virus can be coated on minute gold particles, andthese coated particles are “shot” into biological tissues such ashemangiomas and melanoma under high pressure. An example of the genegun-based method is described for DNA based vaccination of cattle byLoehr B. I. et. al. J. Virol. 2000, 74:6077-86.

In one embodiment, the compositions described herein can be administereddirectly by injection. If the solid tumors and hemangiomas areaccessible by injection, the chimeric VEGF-binding protein, expressionvector, and/or viral vector can be administered by injection directly tothe tumor mass as a pharmaceutical formulation. The preferredformulation is also sterile saline or Lactated Ringer's solution.Lactated Ringer's solution is a solution that is isotonic with blood andintended for intravenous administration.

In the treatment and prevention of diabetic retinopathy and wet maculardegeneration, the present invention, which is much smaller then otherVEGF-binding protein in market and in clinical trials, can be applied tothe eye by injection as a pharmaceutical formulation. The injectiondirectly introduces the chimeric VEGF-binding protein into the vitreoushumor. In one embodiment, the invention compositions can be formulatedas an eye drop solution for direct application on the eyes.

In addition to topical therapy, the compositions described herein canalso be administered systemically in a pharmaceutical formulation.Systemic routes include but are limited to oral, parenteral, nasalinhalation, intratracheal, intrathecal, intracranial, and intrarectal.The pharmaceutical formulation is preferably a sterile saline orlactated Ringer's solution. For therapeutic applications, thepreparations described herein are administered to a mammal, preferably ahuman, in a pharmaceutically acceptable dosage form, including thosethat may be administered to a human intervenously as a bolus or bycontinuous infusion over a period of time, by intramuscular,intraperitoneal, intracerebrospinal, subcutaneous, intra-arterial,intrasynovial, intrathecal, oral, topical, or inhalation routes. Apreferred embodiment is the intramuscular injection of AAV viral vectorsencoding a chimeric VEGF protein and/or its variant forms. Thecompositions described herein are also suitably administered byintratumoral, peritumoral, intralesional or perilesional routes, toexert local as well as systemic effects. The intraperitoneal route isexpected to be particularly useful, for example, in the treatment ofovarian tumors. For these uses, additional conventional pharmaceuticalpreparations such as tablets, granules, powders, capsules, and spraysmay be preferentially required. In such formulations furtherconventional additives such as binding-agents, wetting agents,propellants, lubricants, and stabilizers may also be required. In oneembodiment, the therapeutic compositions described herein are formulatedin a cationic liposome formulation such as those described forintratracheal gene therapy treatment of early lung cancer (Zou Y. et.al., Cancer Gene Ther. 2000 May; 7(5):683-96). The liposome formulationsare especially suitable for aerosol use in lung cancer patients. VectorDNA and/or virus can be entrapped in ‘stabilized plasmid-lipidparticles’ (SPLP) containing the fusogenic lipiddioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol %) ofcationic lipid, and stabilized by a polyethyleneglycol (PEG) coating(Zhang Y. P. et. al. Gene Ther. 1999, 6:1438-47). Other techniques informulating expression vectors and virus as therapeutics are found in“DNA-Pharmaceuticals: Formulation and Delivery in Gene Therapy, DNAVaccination and Immunotherapy” by Martin Schleef (Editor) December 2005,Wiley Publisher, and “Plasmids for Therapy and Vaccination” by MartinSchleef (Editor) May 2001, are incorporated herein as reference. In oneembodiment, the dosage for viral vectors is 10⁽⁶⁾ to 1×10⁽¹⁴⁾ viralvector particles per application per patient.

The route of administration, dosage form, and the effective amount varyaccording to the potency of the chimeric VEGF receptor proteins,expression vectors and viral vectors, their physicochemicalcharacteristics, and according to the treatment location. The selectionof proper dosage is well within the skill of an ordinarily skilledphysician. Topical formulations can be administered up to four-times aday.

In one embodiment, dosage forms include pharmaceutically acceptablecarriers that are inherently nontoxic and nontherapeutic. Examples ofsuch carriers include ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts, orelectrolytes such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, and polyethylene glycol. Carriers for topical or gel-basedforms of chimeric protein include polysaccharides such as sodiumcarboxymethylcellulose or methylcellulose, polyvinylpyrrolidone,polyacrylates, polyoxyethylene-polyoxypropylene-block polymers,polyethylene glycol and wood wax alcohols. For all administrations,conventional depot forms are suitably used. Such forms include, forexample, microcapsules, nano-capsules, liposomes, plasters, inhalationforms, nose sprays, sublingual tablets, and sustained releasepreparations. For examples of sustained release compositions, see U.S.Pat. No. 3,773,919, EP 58,481A, U.S. Pat. No. 3,887,699, EP 158,277A,Canadian Patent No. 1176565, U. Sidman et al., Biopolymers 22:547 (1983)and R. Langer et al., Chem. Tech. 12:98 (1982). The chimeric proteinwill usually be formulated in such vehicles at a concentration of about0.1 mg/ml to 100 mg/ml and the viral vector should be in the range of10⁽⁶⁾ to 1×10⁽¹⁴⁾ viral vector particles per application per patient.

In one embodiment, other ingredients may be added to pharmaceuticalformulations, including antioxidants, e.g., ascorbic acid; low molecularweight (less than about ten residues) polypeptides, e.g., polyarginineor tripeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids, such as glycine, glutamic acid, aspartic acid, or arginine;monosaccharides, disaccharides, and other carbohydrates includingcellulose or its derivatives, glucose, mannose, or dextrins; chelatingagents such as EDTA; and sugar alcohols such as mannitol or sorbitol.

In one embodiment, the pharmaceutical formulation to be used fortherapeutic administration must be sterile. Sterility is readilyaccomplished by filtration through sterile filtration membranes (e.g.,0.2 micron membranes). The chimeric VEGF receptor protein ordinarilywill be stored in lyophilized form or as an aqueous solution if it ishighly stable to thermal and oxidative denaturation. The pH of thechimeric VEGF receptor protein preparations typically will be about from6 to 8, although higher or lower pH values may also be appropriate incertain instances.

For the prevention or treatment of angiogenic disease or disorder, theappropriate dosage of chimeric VEGF receptor protein and/or viralvectors will depend upon the type of disease or disorder to be treated,the severity and course of the disease, whether the chimeric VEGFreceptor proteins are administered for preventative or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the chimeric VEGF receptor protein and/or viral vectors and thediscretion of the attending physician. The chimeric VEGF receptorprotein and/or viral vectors are suitably administered to the patient atone time or over a series of treatments. For purposes herein, the“therapeutically effective amount” of a chimeric VEGF receptor proteinor viral vector is an amount that is effective to either prevent, lessenthe worsening of, alleviate, or cure the treated condition, inparticular that amount which is sufficient to reduce or inhibit theproliferation of vascular endothelium in vivo.

This invention is further illustrated by the following example whichshould not be construed as limiting. The contents of all referencescited throughout this application, as well as the figures and tables areincorporated herein by reference.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean±1%.

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

EXAMPLE

VEGF binding proteins, in the form of mAb's, soluble VEGF receptorextracellular domains, or heterologous constructs derived fromsubdomains of VEGF receptors (referred to as VEGF-Traps) have been shownto exhibit potent anti-tumor effects in preclinical (1, 2) and clinicalstudies (3). Studies to date have largely utilized direct systemicprotein delivery of these proteins. Because of the requirement forsustained delivery of anti-angiogenic agents to maintain an anti-tumoreffect, there is interest in investigating the use of gene therapy basedapproaches for delivering VEGF binding proteins in order to achievesustained systemic delivery of protein. In a prior report, (2) it wasnoted that systemically delivered adenoviruses could secrete a highlevel of soluble VEGF receptor extracellular domains and control thegrowth of several different human tumor xenografts in vivo. Thisadenovirus based system is less favorable for clinical use secondary toissues of immune mediated reduction in transgene expression andpotential for organ specific (e.g. liver) toxicity after systemic virusdelivery. Because of these limitations the strategy of delivering theseproteins using more clinically acceptable vectors delivered viaintramuscular injection was explored.

To this end, a smaller, neutrally charged VEGF binding protein VEGF-trap3 (VT3) was designed and expressed, more appropriate for the smallerpackaging capability of adeno-associated virus, as well as those withenhanced charge properties compared to the original VEGF-Trap describedby Holash et al. (1) This modification entailed removing one of twobasic Ig VEGF binding domain in the parental VEGF-Trap1 (VT1) proteins.It was found that this modified trap retained a similar binding affinityto VEGF compared to VT1. The modified VEGF-Trap3 (VT3) yieldedsignificantly higher levels of serum protein when delivered via an AAVvector using a direct intramuscular delivery technique. The uniquefeatures of this Trap make it particularly suitable for gene therapyapproaches for anti-VEGF anti-angiogenic therapeutic strategies.

Materials and Methods. VEGF Binding Proteins

Purified VEGF-Trap 1 (VT1) was obtained as a gift from RegeneronPharmaceuticals. Recombinant human Flt-1/Fc chimera (R&D System 321-FL),recombinant human KDR/Fc chimera (R&D System 357-KD) and recombinanthuman IgG₁ Fc (R&D System 110-HG) were purchased from R&D Systems.

Engineering VEGF-Trap1 and VEGF-Trap3 Encoding AAV Vectors

In order to construct an AAV vector encoding VEGF-Trap 1 (VT1), fusionPCR of transgene encoding oligos was used to create a full lengthVEGF-Trap construct encoding the signal sequence of VEGF-receptor 1(Amino acids 1-26 of Genbank Accession No.: BCO39007, SEQ. ID. No.: 1)fused directly to the second Ig domain (Amino acids 129-231 of GenbankAccession No.: BCO39007, SEQ. ID. No.: 1) fused directly to the third Igdomain of VEGF-Receptor 2 (amino acids 226-327 of Genbank Accession No.:AF035121, SEQ. ID. No.: 9) fused directly to the huIgG1 (amino acids247-473 of Genbank Accession No.: BC092518) as described Holash et al.(Holash et al., 2002).

Vegf-Trap 3 (VT3) was similarly constructed by attaching the second Igdomain of Flt-1 to Fc-huIgG₁. The final construct encodes the signalsequence of VEGF-receptor 1 (Amino acids 1-26 of Genbank Accession No.:BCO39007, SEQ. ID. No.: 1) the second Ig domain (Amino acids 129-231 ofGenbank Accession No.: BCO39007, SEQ. ID. No.: 1) fused directly to thehuIgG1 (amino acids 247-473 of Genbank Accession No.: BC092518). Finalconstructs encoding both VT1 and VT3 were sequenced in both sense andanti-sense strands to confirm correct sequence and orientation.

Production of recombinant AAV vectors encoding VEGF-Trap1 and VEGF-Trap3 was performed by triple transfection as described previously (4).Briefly, the pAAV-VEGF-Trap vector plasmids were co-transfected with AAVhelper plasmid pLT-RC02 (Jeng-Shin Lee and Richard Mulligan,unpublished) and adenovirus helper miniplasmid pHGTI-Adeno1 (John Grayand Richard Mulligan, unpublished) into 293 cells. 48 hours aftertransfection, the cells were harvested and lysed. After Bezonasetreatment and removal of cell debris by low speed centrifugation, therecombinant AAV particles were purified by iodixanol density gradient.The 40% iodixanol fraction containing rAAV particles was recovered,dialyzed extensively against PBS and titered by dot-blot hybridizationusing the CMV promoter as the probe.

pLT-RC02 encodes hybrid Rep proteins from AAV serotypes 1 and 2, and Capproteins from serotype 1. When used in the triple tranfection productionsystem, pLT-RC02 plasmid packages AAV particles at a comparable levelrelative to that obtained with AAV serotype 2 Rep/Cap expressionplasmid. But the resultant vector particles transduced skeletal musclesat significantly higher efficiency than regular serotype 2 vectors(Jeng-Shin Lee and Richard Mulligan, unpublished) as reported previouslyfor AAV serotype 1 vectors.

Protein Purification of VEGF-Trap 3.

Stable production of VEGF-Trap 1 and VEGF-Trap 3 in 293T cell wasachieved by infection with lentiviruses encoding the respectiveVEGF-Trap proteins. Stable production of the VEGF-Traps was confirmed byELISA. Serum free Dulbecco's Modified Eagle Medium (Gibco 11965-092)conditioned media from infected cells was subsequently pooled andsubjected to Protein A affinity chromatography.

NProtein A Sepharose 4 Fast Flow (Amersham Biosciences 17-5280-04)columns were prepared according to manufacturer's instructions. In orderto ensure proper ionic strength, serum free conditioned media wassupplemented with 3.3M NaCl prior to being applied to the column.Samples were loaded overnight at 4° C. then column was washed with 5 bedvolumes of 50 mM Tris, 150 mM NaCl and 0.1% Tween-20 pH 8.0. VEGF-Trapproteins were eluted with 1-3 bed volumes 0.1M Citrate pH3 andneutralized with 2M Tris pH8.5. Elutes were dialyzed againstphosphate-buffered saline (PBS) (Gibco) in 10,000 MWCO dialysiscassettes (Pierce 66380) at 4° C., overnight. Finally the samples wereconcentrated by Amicon Ultra low binding centrifugal filter devices(Millipore UFC901008) and purity was assessed by SDS PAGE and Coomassiestaining

Protein Characterization—Glycosylation and Western Blot Analysis

Purified protein samples were subjected to SDS PAGE (12% gel, Life Gels,NH21-012) at 100V for 45 minutes then the gel was transferred in a wetelectrotransfer system at a constant current of 35 mA for 2 hours.Following the transfer, supported nitrocellulose membrane (Bio-Rad162-0095) was blocked with 10% Non-Fat Dried Milk in PBS-0.1% Tween-20at room temperature for 1 hour. Membrane was probed with anti-Flt-1 (R&DSystem AF321 or Imclone) primary and HRP conjugated anti-goat-IgGsecondary antibody or with HRP-conjugated primary antibody againsthuman-IgG-Fc (Sigma A0170) diluted in PBS-0.1% Tween-20. Blots weredeveloped by using ECL-Plus (Amersham Biosciences RPN2132) HRP substrateand Kodak Biomax Chemiluminescence Film (Kodak 178 8207).

In order to remove all N and O-linked carbohydrates, protein sampleswere denatured at 100° C. for 5 minutes then incubated with PNGase F,α-2(3,6,8,9)Neuraminidase, O-Glycosidase, β(1-4)-Galactosidase andβ-N-Acetylglucosaminidase enzymes (Sigma, E-DEGLY) overnight at 37° C.Extend of the glycosylation was assessed by SDS-PAGE of deglycosylatedand non-deglycosylated protein samples. Following the electrophoresisthe gel was Coomassie stained (Gradipore, Gradiflash SG-010) for 5 h atroom temperature then distained with 6% Acetic Acid at room temperaturefor 20 hours.

Binding Affinity Measurement

Binding affinities of Vegf-Trap 3, recombinant VEGF-Trap 1 (gift ofRegeneron pharmaceuticals), recombinant human Flt-1/Fc chimera (R&DSystem 321-FL), recombinant human KDR/Fc chimera (R&D System 357-KD) andrecombinant human IgG₁ Fc (R&D System 110-HG) were measured by ELISA(R&D System DVE00) as previously described by Holash et al. (1.)

Serum VEGF-Trap detection—VEGF-Trap ELISA

Immulon-4 HBX plates are coated with 25 ng/well human Vegf165 (R&DSystem 293 VE) overnight at 4° C. Plates are blocked with 3% Non-FatDried Milk in PBS-0.1% Tween-20 at room temperature for 1 hour.

Standards and serum samples are diluted in PBS-10% mouse serum-0.1%Tween-20 solution, applied to the plate and incubated at roomtemperature for 1 hour. Plates are washed with PBS-0.1% Tween-20 fivetimes. Diluted (1:5000), HRP-conjugated detection antibody againsthuman-IgG-Fc (Sigma A0170) is added to the wells. After 1 hourincubation at room temperature plates are washed with PBS-0.1% Tween-20.O.D. is read at 405 nm within 10 min of adding HRP-substrate (KPL50-66-06).

Pharmacokinetic Analysis—BALB/c Injection with Protein

BALB/c and C57Bl/6 mice (20-25 g) were injected with 100 ug/animalVegf-Trap 3 or VEGF-Trap 1 s.c. The mice were bled at 1, 6, 24, 48, 72,96, 120, 144 hours after injection. The levels of Vegf-Trap3 andVEGF-Trap 1 were measured by ELISA.

AAV-Delivery

Mice were obtained from Jackson Labs (Bar Harbor, Me.) and maintained ina pathogen-free animal facility at Harvard Institute of Medicine,Boston, Mass. Animal studies were approved by the Standing Committee onAnimals of Harvard Medical School.

Tumor Growth Studies

Animal studies were carried out in the animal facility at ChildrensHospital, Boston, Mass., in accordance with federal, local andinstitutional guidelines. Immunocompetent, male C57B16/J mice (JacksonLabs, Bar Harbor, Me.) 7-9 weeks of age were used. Lewis Lung Carcinoma(LLC) cells (American Type Culture Collection, Rockville, Md.) weregrown and maintained as described earlier (5) A suspension of 5×10⁶tumor cells in 0.1 ml DMEM was injected s.c. into the dorsa of mice atthe proximal midline.

Mice were weighed and tumors were measured every 3 to 5 days in twodiameters with a dial caliper. Volumes were determined using a²×b×0.52formula, where a is the shortest and b is the longest diameter. When theaverage tumor volume reached approximately 100 mm³ mice were randomizedinto seven treatment groups (n=4). VEGF-Trap proteins and the placebocontrol were administered twice a week subcutaneously. Upon completionof the experiment mice were euthanized by CO₂ asphyxiation. Tumors werefixed in 10% buffered formalin (Fisher Scientific) overnight at 4° C.then processed for immunohistochemistry as described previously. (6)

Results

Two different expression cassettes were ligated into AAV vectors tocreate AAV-VT1 encoding VEGF-Trap 1 (Holash et al, 2002) andAAV-VEGF-Trap 3 as shown in FIG. 1. 293T cells stably expressing theseconstruct yielded purified proteins from 293 cell conditioned media ofthe predicted (VT1-48.7 kDa, VT3-37.4 kDa) size when analyzed underdeglycosylating conditions (FIG. 2-lane 3-VT3, lane 6-VT1). Furthermore,similar levels of protein were produced when 293T cells were infectedwith AAV virus as assessed by Elisa. As shown in FIG. 3, there was asimilar dose dependent rise in concentration of both VEGF-Trap 1 andVEGF-Trap 3 with increasing multiplicity of infection. The measured meanconcentrations of these two proteins were within 20% of each other andthis was not different statistically.

A binding affinity analysis (FIG. 4) revealed that both VT1 and VT3 hada superior binding affinity for VEGF compared to the full-lengthVEGF-Receptor 1 and Receptor 2 extracellular domains. VT1 bindingaffinity was estimated at 1.8 pM and that of VT3 at 7.3 pM. VEGF-R1extracellular domain Ig fusion demonstrated a binding affinity of 25.9pM and VEGF-R2 extracellular domain, 92.3 pM.

Following subcutaneous deposition of 100 μg of purified protein into theflanks of nude mice, a slightly higher peak serum concentration ofVEGF-Trap 1 was achieved (FIG. 5) (27.5 μg/ml versus 14 μg/ml) althoughboth proteins were still present in the serum at 120 hours postinjection. This finding was in marked contrast to the levels of proteindetected after AAV based gene delivery into skeletal muscle in which wenoted that VEGF-Trap 3 had a markedly improved pharmacokinetic profilecompared to VEGF-Trap 1. For this experiment, mice were injectedintramuscularly with 1.25 (VT3)-1.85 (VT1) Ell AAV particles in a totalvolume of 50 μl. Serial serum ELISA measurements revealed that while AAVVT3 protein could be detected at approximately 10 μg/ml for >120 days.AAV VT1 protein was present in the serum at <0.1 μg/ml for the duration(120 days) of analysis (FIG. 6). These data showed that AAV-mediateddelivery of VT3 protein result in a sustained and longer term expressionof VT3 in vivo, and that is significantly more effective than the AAVmediated delivery of VT1.

The level of VEGF-Trap 3 required to inhibit tumor growth wasdetermined. This was performed using the angiogenesis sensitivesubcutaneous Lewis lung carcinoma tumor model. Animals were injected inthe flank with 5E5 cells. 7 days later biweekly treatment with VEGF-Trap3 was performed. As observed in FIG. 7, there was a dose dependentinhibition of LLC tumor growth, while animals receiving as low 1mg/kg/dose showed growth inhibition compared to PBS treated mice, thebest growth inhibition was observed with biweekly dosing of 4 mg/kg.Serum VEGF-Trap 3 levels were also measured at two points during thestudy. At Day 6, 72 hours after the second treatment dose, the averageVEGF-Trap 3 levels were 3.6, 10.1, and 62 μg/ml for the 1, 2, and 4mg/kg dosing regimens respectively. At day 14, 96 hours after the 4^(th)dose, VEGF-Trap levels were 0.7, 5.9, and 30.2 μg/ml for the 1, 2, and 4mg/kg dosing regimens respectively.

Gene based delivery of anti-angiogenic proteins offers theoreticaladvantages over repeated protein administrations of such proteins. Acontinuous and persistent level of angiogenesis inhibition is requiredto optimize the efficacy of this therapy. Of the various modalities ofgene delivery currently available, AAV based transduction of skeletalmuscle is considered a relatively safe modality and clinical trialsusing this modality are underway(4). However, after gene delivery, avariety of factors may influence the pharmacokinetic profile of thedelivered protein. The original VEGF-Trap protein was designed to removepositively charged motifs compared to full length VEGF receptorextracellular domains and thereby reduce interactions with negativelycharged proteoglycans in the extracellular matrix following subcutaneousdeposition. In the initial efforts to develop gene therapy vectorscapable of delivering a sufficiently high serum level of anti-angiogenicactivity by using the VEGF-Trap 1, it was found (FIG. 6) that levels ofVEGF-Trap 1 secreted by AAV vectors after IM injection were very low.The VEGF-trap 1 was modified by removing the 3^(rd) Ig domain of theVEGF-Receptor 2, one of two VEGF binding motifs present in VEGF-Trap 1.This modification further reduced the size and charge of the protein.Following AAV based transduction of skeletal muscle, the levels ofVEGF-Trap 3 achieved were 2 logs greater than those could be achievedfrom a VEGF-Trap 1 expressing vector.

The references cited herein and throughout the specification areincorporated herein by reference.

REFERENCES

-   1. Holash, J., Davis, S., Papadopoulos, N., Croll, S. D., Ho, L.,    Russell, M., Boland, P., Leidich, R., Hylton, D., Burova, E., Ioffe,    E., Huang, T., Radziejewski, C., Bailey, K., Fandl, J. P., Daly, T.,    Wiegand, S. J., Yancopoulos, G. D., and Rudge, J. S. VEGF-Trap: a    VEGF blocker with potent antitumor effects. Proceedings of the    National Academy of Sciences of the United States of America, 99:    11393-11398, 2002.-   2. Kuo, C. J., Farnebo, F., Yu, E. Y., Christofferson, R.,    Swearingen, R. A., Carter, R., von Recum, H. A., Yuan, J., Kamihara,    J., Flynn, E., D'Amato, R., Folkman, J., and Mulligan, R. C.    Comparative evaluation of the antitumor activity of antiangiogenic    proteins delivered by gene transfer. Proceedings of the National    Academy of Sciences of the United States of America, 98: 4605-4610,    2001.-   3. Hurwitz, H., Fehrenbacher, L., Novotny, W., Cartwright, T.,    Hainsworth, J., Heim, W., Berlin, J., Baron, A., Griffing, S.,    Holmgren, E., Ferrara, N., Fyfe, G., Rogers, B., Ross, R., and    Kabbinavar, F. Bevacizumab plus irinotecan, fluorouracil, and    leucovorin for metastatic colorectal cancer. New England Journal of    Medicine, 350: 2335-2342, 2004.-   4. Flotte, T. R., Brantly, M. L., Spencer, L. T., Byrne, B. J.,    Spencer, C. T., Baker, D. J., and Humphries, M. Phase I trial of    intramuscular injection of a recombinant adeno-associated virus    alpha 1-antitrypsin (rAAV2-CB-hAAT) gene vector to AAT-deficient    adults. Human Gene Therapy, 15: 93-128, 2004.-   5. Kisker, O., Becker, C. M., Prox, D., Fannon, M., D'Amato, R.,    Flynn, E., Fogler, W. E., Sim, B. K., Allred, E. N.,    Pirie-Shepherd, S. R., and Folkman, J. Continuous administration of    endostatin by intraperitoneally implanted osmotic pump improves the    efficacy and potency of therapy in a mouse xenograft tumor model.    Cancer Research, 61: 7669-7674, 2001.-   6. Tjin Tham Sjin, R. M., Satchi-Fainaro, R., Birsner, A. E.,    Ramanujam, V. M., Folkman, J., and Javaherian, K. A 27-amino-acid    synthetic peptide corresponding to the NH2-terminal zinc-binding    domain of endostatin is responsible for its antitumor activity.    Cancer Research, 65: 3656-3663, 2005.

1. A chimeric VEGF-binding protein comprising a first portion, the firstportion consisting of amino acids 129-231 of the flt-1 tyrosine kinasereceptor (SEQ ID NO:1), and a second portion comprising an Fc region ofimmunoglobulin G1.
 2. The chimeric VEGF-binding protein of claim 1,further comprising the signal peptide of the flt-1 tyrosine kinasereceptor (SEQ ID NO:5).
 3. The chimeric VEGF-binding protein of claim 1wherein said Fc region of immunoglobulin G1 is a human Fc region ofimmunoglobulin G1.
 4. The chimeric VEGF-binding protein of claim 1,wherein the Fc region of immunoglobulin G1 comprises amino acids 247-473of IgG1 (Genbank accession # BC092518).
 5. The chimeric VEGF-bindingprotein of claim 1 wherein said Fc region comprises a reducedimmunogenic derivative of an Fc region of immunoglobulin G1.
 6. Apharmaceutical composition comprising the chimeric VEGF-binding proteinof claim 1 and a pharmaceutically acceptable carrier.
 7. An isolatedpolynucleotide encoding the chimeric VEGF-binding protein of claim
 1. 8.A recombinant vector comprising the polynucleotide of claim
 7. 9. Therecombinant vector of claim 8, wherein the recombinant vector is anexpression vector that is compatible with a protein expression systemusing host cells selected from the group consisting of: mammalian cells;insect cells; yeast cells; bacterial cells; and plant cells.
 10. A hostcell comprising the expression vector of claim
 9. 11. The recombinantvector of claim 8, wherein the vector is a viral vector.
 12. The viralvector of claim 11, wherein the vector is an adeno-associated virus(AAV) vector.
 13. The viral vector of claim 11, wherein the vector is alentivirus vector.
 14. A method of treating an angiogenic disease ordisorder, comprising administering to a subject in need thereof a vectorcomprising the polynucleotide of claim
 7. 15. A method of treating anangiogenic disease or disorder, comprising administering to a subject inneed thereof a pharmaceutical composition comprising a chimericVEGF-binding protein, wherein the chimeric VEGF-binding proteincomprises an immunoglobulin-like domain 2 of a vascular endothelialgrowth factor receptor and an Fc region of immunoglobulin G1 or areduced immunogenic derivative of a said Fc region.
 16. A method ofproducing a chimeric VEGF-binding protein, the method comprisingintroducing a recombinant vector of claim 8 into an isolated host cell,growing the cell under conditions permitting the production of thechimeric protein and recovering the chimeric protein so produced.
 17. Amethod of treating an angiogenic disease or disorder, comprisingadministering to a subject in need thereof a pharmaceutical compositionof claim 6.